A single-tube six-colour flow cytometry screening assay for the detection of minimal residual disease in myeloma

Detection of minimal residual disease (MRD) in myeloma is highly predictive of early relapse following treatment. A number of different approaches to myeloma MRD detection are available; these vary widely in sensitivity and cost. Although several studies suggest that detection of neoplastic plasma cells above a level of 0.01% is clinically relevant,1, 2, 3, 4, 5, 6 MRD studies in B-cell chronic lymphocytic leukemia (CLL) comparing different approaches have demonstrated that methods such as consensus-primer immunoglobulin heavy-chain (IgH) PCR and assessment of light-chain restriction by flow cytometry can detect low levels of disease but the sensitivity is highly variable. Such approaches are least informative when large numbers of polyclonal B cells are present as is typical after high-dose therapy and stem cell transplantation. In this setting, false-negative results may be generated in cases with up to 5% residual disease. Therefore, approaches that can directly quantify the proportion of CLL cells, using either CLL-specific flow cytometry assays or real-time quantitative allele-specific oligonucleotide (RQ-ASO) IgH-PCR are recommended for MRD detection as the sensitivity is not impaired by the presence of polyclonal B cells.7, 8

In both CLL and myeloma, flow cytometric assessment of MRD may be preferable in practice to RQ-ASO IgH-PCR since a clinically relevant limit of MRD detection can be achieved using a standard antibody panel whereas RQ-ASO IgH-PCR requires the cost and labour-intensive development of patient-specific primers. However, quantitative MRD detection of myeloma plasma cells by flow cytometry is relatively complicated compared to CLL flow cytometric MRD analysis. Myeloma MRD flow cytometry requires at least two markers for plasma cell identification (CD38 and either CD45 or CD138, preferably all three) and some combination of several additional markers to detect phenotypic abnormality including CD19, CD20, CD27, CD28, CD45, CD56 and CD117.4, 5, 6, 9, 10 As most of the myeloma-associated phenotypic plasma cell abnormalities can also be detected at low levels in healthy individuals, assessment of immunoglobulin light-chain restriction (cytoplasmic κ and λ) is also informative.

Six-colour flow cytometry offers the opportunity to combine the gating markers CD38/CD138/CD45 with clonality assessment and basic immunophenotypic characterization simultaneously in a smaller number of individual assays than three-/four-colour flow cytometry. This could potentially increase the sensitivity of the method through coincident multiparameter analysis and also reduce the cost and time required to generate a result. The aim of this study was to determine the efficacy of a single-tube six-colour analysis for providing quantitative MRD detection in myeloma patients.

Bone marrow aspirate samples were analysed from 62 patients, median age 61.8 years (43.2–92.1), 41 men and 21 women, with myeloma diagnosed according to standard criteria.11 The samples were obtained following treatment with induction therapy (n=44), at day 100 after high-dose consolidation therapy (n=11) or after the end of therapy (n=7) according to the Myeloma IX trial protocol (http://www.ukmf.org.uk/documents/myeloma9.pdf). All study patients consented to bone marrow investigation and flow cytometric studies were performed as part of the trial protocol.

Up to 1 ml of whole marrow aspirate in ethylenediamine tetraacetic acid (EDTA) was washed twice in 10 ml FACSFlow (BD Biosciences, Oxford, UK) containing 0.1% bovine serum albumin (Sigma, Gillingham, UK), and the cells resuspended to a concentration of 20 × 106 per ml. Precisely 50 μl of washed cells were incubated with 10 μl each of optimally titrated antibody CD19 PE, CD38 PE-Cy7, CD138 APC and CD45 APC-Cy7 (all from BD Biosciences) for 20 min at 4°C in the dark. Antibody cocktails were prepared daily to avoid degradation of Cy7 tandem conjugates. The cells were then fixed and permeabilized using the INTRASure kit (BD Biosciences) and incubated with 10 μl each of Kappa FITC and Lambda PE-Cy5 (both in-house conjugates) for 20 min at 4°C in the dark and then washed. Then 200 000 events were acquired on a FACSCanto flow cytometer and the results analysed using FACSDiva software (BD Biosciences) with a minimum population size of 20 events.

The gating strategy is shown in Figure 1. The information derived from this assay was: (i) plasma cells as a percentage of total leukocytes; (ii) plasma cell immunophenotype with respect to CD19 and CD45 expression; (iii) cytoplasmic κ/λ expression by CD19− and CD19+ plasma cells; (iv) B cells as a percentage of total leukocytes; (v) the percentage of CD38strong CD45dim B progenitors and CD38+/−CD45strong mature B cells; and (vi) κ/λ expression by B progenitors and mature B cells. The surface or cytoplasmic κ/λ ratio was classified as abnormal if lower than 0.5:1 or greater than 3:1; a population was classified as monoclonal if >95% of the cells expressed either κ or λ. Patients with less than 5% plasma cells by morphology were classified as being in morphological remission and subclassified as MRD-positive if neoplastic plasma cells represented between 0.01 and 5% of leukocytes or MRD-negative if neoplastic plasma represented <0.01% of leukocytes in a representative aspirate sample. Representative aspirate samples were defined as those containing bone marrow elements (erythroid and myeloid progenitors, megakaryocytes, B progenitors and/or normal plasma cells) identified by morphology and/or flow cytometry. MRD status was classified independently of the immunofixation status because flow cytometry is more sensitive5 and it can take several months for the serum paraprotein to disappear after maximum response.12 Detection of residual disease was compared with our previously reported three-tube three-colour approach, which uses CD38/CD45/CD138 to quantify plasma cells and identify optimal CD38/CD45 gates.5 In this method, these gates are then used with CD3 (negative control) to assess CD19 and CD56 expression on plasma cells and cases classified as having residual disease if >10% of total plasma cells are either CD19− and/or CD56+.

Figure 1
figure1

Analysis of the single-tube six-colour assay. The figure shows the single FACSDiva analysis page subdivided into the major analysis components. (a) Plasma cells are gated according to CD38/CD138 (A1), FSC/SSC (A2) and CD38/CD45 (A3) characteristics and the phenotype with respect to CD19 and CD45 expression is determined (A4). (b) Cytoplasmic k/l expression is determined on gated CD19+ plasma cells (B1) and CD19- plasma cells (B2), in this case showing polyclonal CD19+ and monoclonal CD19 plasma cells. Minimal residual disease (MRD) was considered to be present when a discrete population of CD19 plasma cells representing more than 0.01% of leukocytes with restricted cytoplasmic light-chain expression were detected, that is more than 95% of the events in B1 were either in the upper left sector or the lower right sector. (c) B cells are identified by CD19/SSC (C1) and FSC/SSC (C2) characteristics and subdivided into B progenitors with strong CD38 and weak/moderate CD45, and mature B cells with strong CD45 (C3). (d) Surface k/l expression is assessed on gated mature B cells (D1) and B progenitors (D2). (e) Enumeration of leukocytes and nucleated red cells according to CD45/SSC characteristics. This allows enumeration of plasma cells and B cells combined with basic immunophenotyping and clonality in a single test.

In our six-colour assay, MRD was considered to be present when a discrete population of light chain restricted CD19− plasma cells representing more than 0.01% of leukocytes was detected. MRD as defined in this way was demonstrable in 71% (44/62) of samples from patients during or immediately after treatment. In the remaining 18 cases, it was not possible to determine MRD levels but it was possible to quantify the level of total plasma cells, and also to determine the proportion of CD19− plasma cells and the κ/λ ratio. This information can be used to determine the upper limit of residual disease detection. It is not possible with this single-tube assay to determine whether these samples were truly MRD-negative or had very low levels of residual disease. Definitive assessment of MRD in these samples would therefore require extended immunophenotypic analysis. The presence of MRD was clearly associated with the total plasma cell percentage as it was demonstrable in 94% (17/18) of samples containing more than 1% plasma cells, in 67% (23/34) of samples containing 0.1–1% plasma cells, and in 40% of cases (4/10) containing 0.01–0.1% plasma cells as a percentage of total leukocytes (see Figure 2).

Figure 2
figure2

Quantitative detection of residual disease using the single-tube six-colour assay. Detection of minimal residual disease (MRD), defined as the presence of a discrete population of CD19− plasma cells with restricted immunoglobulin light-chain expression, was possible in 71% of patients (44/62). The assay has a limit of detection of 0.01% of total leukocytes. This limit of detection is achieved in samples devoid of normal plasma cells. If normal plasma cells are present the assay provides a qualitative result identifying the upper limit of residual disease.

In all cases, the single-tube six-colour assay produced equivalent results to the four-tube three-colour analysis. There was no increase in specificity or sensitivity of this assay over our current approach, which has a limit of detection of 1 abnormal cell in 10 000 normal leukocytes in patients undergoing or immediately after therapy. It is important to note, however, that the three-colour approach only achieves this limit of detection because polyclonal (normal) CD19− and/or CD56+ plasma cells, which limit the ability to enumerate neoplastic plasma cells, are virtually absent in patients undergoing or immediately after therapy. Polyclonal CD19−/CD56+ plasma cells become detectable in some patients at least 1 year after therapy and it is likely that the single-tube six-colour assay will be more informative in this setting. This may be particularly important in providing an objective measure of response in trials of maintenance therapy.

There was a 22% reduction in antibody cost in using the single-tube six-colour assay compared to the four-tube three-colour assay.5 We have also previously developed a four-colour assay including clonality assessment:13 similar to the six-colour assay, this did not show an improvement in sensitivity (data not shown) but antibody costs were 2.2-fold higher than the three-colour assay. For high-throughput centres like the HMDS laboratory where over 2000 such assays are performed per year this equates to a potential saving of £70 000 (€100 000 or $140 000) per year in reagent costs when using the single-tube six-colour assay compared to multi-tube four-colour analysis. In addition to the major reduction in cost compared to three-/four-colour analysis, acquisition time was reduced and gating was more straightforward and reproducible as all the relevant markers were present in the same test.

In summary, the single-test six-colour assay provides plasma cell enumeration with a limit of detection of 0.01%. The assay is semiquantitative but, unlike other semiquantitative approaches such as consensus primer IgH-PCR, can directly enumerate the level of neoplastic plasma cells in samples from the majority of patients during and immediately after treatment. This approach is being tested prospectively in parallel with extended immunophenotypic and RQ-ASO IgH-PCR MRD analysis in the UK MRC Myeloma IX trial, although it will be several years before outcome data are available. In addition to MRD detection, this approach also provides detailed information about other cells in the sample, which can be used to assess sample quality and check for the presence of monoclonal B cells, and may therefore be used as a screening test in patients being investigated for monoclonal gammopathy. This approach is faster and less expensive than three-colour and four-colour methods. This relatively simple test offers the potential for most diagnostic flow cytometry laboratories to perform high-sensitivity MRD analysis.

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Acknowledgements

We thank Medical Research Council (MRC), UK and Leukaemia Research Fund (LRF), UK. RMdT and ACR are members of the EuroFlow consortium.

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Correspondence to A C Rawstron.

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de Tute, R., Jack, A., Child, J. et al. A single-tube six-colour flow cytometry screening assay for the detection of minimal residual disease in myeloma. Leukemia 21, 2046–2049 (2007). https://doi.org/10.1038/sj.leu.2404815

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